Signal transduction underlying carbachol-induced contraction of human urinary bladder

The present study was designed to reexamine the muscarinic acetylcholine receptor subtype mediating carbachol-induced contraction of human urinary bladder and to investigate the underlying signal transduction. Based upon the nonselective tolterodine, the highly M-2-selective (R)-4-{2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl}piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M-3-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M-3 receptors. The phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122) (1-10 muM) did not significantly affect carbachol-stimulated bladder contraction. The phospholipase D inhibitor butan-1-ol relative to its negative control butan-2-ol (0.3% each) caused small but detectable inhibition of carbachol-induced bladder contraction. The Ca2+ entry blocker nifedipine (10-100 nM) strongly inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SK&F 96,365) (1-10 muM), an inhibitor of store-operated Ca2+ channels, caused little inhibition. The protein kinase C inhibitor bisindolylmaleimide 1 (1-10 muM) did not significantly affected carbachol-induced bladder contraction. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 muM), an inhibitor of rho-associated kinases, concentration dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of human urinary bladder via M-3 receptors largely depends on Ca2+ entry through nifedipine-sensitive channels and activation of a rho kinase, whereas phospholipase D and store-operated Ca2+ channels contribute only in a minor way. Surprisingly, phospholipase C or protein kinase C do not seem to be involved to a relevant extent.